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Providing evidence for presumed chemolithoautotrophic manganese oxidation remains a major and challenging objective in subsurface microbiology. Here, we report on the dissection of blackish, leathery microbial biofilms discovered in the “Goldene Falk”, a historic copper mine in Northern Bavaria, with mine shafts originating back to the 15 th  centrury. Biogeochemical analysis of the biofilm indicated a notable enrichment of manganese oxides (MnOX), with Mn making up for more than 10% (dry weight) of the deposits. STXM analysis suggested a clear biogenic origin of MnOX  in situ . Characteristic nodules of MnOX with microbial cells attached were also found in aerobic Mn-oxidizing enrichment cultures set up in minimal media in the lab. The biofilms obtained from the mine were also subjected to amplicon and metagenomic sequencing,revealing a vast diversity of presumably chemolithoautotrophic and heterotrophic microbial lineages, including members of the  Pyrinomonadaceae ,  Rhizobiales ,  Methylomirabilaceae  and also lineages within the  Nitrospiraceae  previously reported to be associated with lithotrophic Mn oxidation. We reconstructed &gt;100 high-quality bacterial genomes (MAGs), many of them carrying genomic signatures of biogenic Mn oxidation (albeit non-lithotrophic). We continue to investigate the biofilms, our enrichment cultures and the metagenomic data obtained from the mine for further evidence of possible autotrophic manganese oxidation, the macroscopic leathery biofilm representing a likely habitat for these still enigmatic microbes. Indications for nitrogen and sulfur cycling also ongoing in the biofilms will also be discussed. This research contributes to a better understanding of the yet-enigmatic capacities of the microbiota in man-made subsurface environments.

Investigating Manganese-oxidizing microbial Biofilms in a historic Copper Mine of Upper Frankonia